Fungal and other mycotic pathogens (some of which are described in Human Mycoses, E. S. Beneke, Upjohn Co.:Kalamazoo, Mich., 1979; Opportunistic Mycoses of Man and Other Animals, J. M. B. Smith, CAB International:Wallingford, UK, 1989; and Scrip's Antifungal Report, by PJB Publications Ltd, 1992) are responsible for a variety of diseases in humans, animals, and plants ranging from mycoses involving skin, hair, or mucous membranes, such as, but not limited to, Aspergillosis, Black piedra, Candidiasis, Chromomycosis, Cryptococcosis, Onychomycosis, or Otitis externa (otomycosis), Phaeohyphomycosis, Phycomycosis, Pityriasis versicolor, ringworm, Tinea barbae, Tinea capitis, Tinea corporis, Tinea cruris, Tinea favosa, Tinea imbricata, Tinea manuum, Tinea nigra (palmaris), Tinea pedis, Tinea unguium, Torulopsosis, Trichomycosis axillaris, White piedra, and their synonyms, to severe systemic or opportunistic infections, such as, but not limited to, Actinomycosis, Aspergillosis, Candidiasis, Chromomycosis, Coccidioidomycosis, Cryptococcosis, Entomophthoramycosis, Geotrichosis, Histoplasmosis, Mucormycosis, Mycetoma, Nocardiosis, North American Blastomycosis, Paracoccidioidomycosis, Phaeohyphomycosis, Phycomycosis, pneumocystic pneumonia, Pythiosis, Sporotrichosis, and Torulopsosis, and their synonyms, some of which may be fatal. Known fungal and mycotic pathogens include, but are not limited to, Absidia spp., Actinomadura madurae, Actinomyces spp., Allescheria boydii, Alternaria spp., Anthopsis deltoidea, Apophysomyces eleqans, Arnium leoporinum, Aspergillus spp., Aureobasidium pullulans, Basidiobolus ranarum, Bipolaris spp., Blastomyces dermatitidis, Candida spp., Cephalosporium spp., Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Coccidioides immitis, Conidiobolus spp., Corynebacterium tenuis, Cryptococcus spp., Cunninghamella bertholletiae, Curvularia spp., Dactylaria spp., Epidermophyton spp., Epidermophyton floccosum, Exserophilum spp., Exophiala spp., Fonsecaea spp., Fusarium spp., Geotrichum spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Madurella spp., Malassezia furfur, Microsporum spp., Mucor spp., Mycocentrospora acerina, Nocardia spp., Paracoccidioides brasiliensis, Penicillium spp., Phaeosclera dematioides, Phaeoannellomyces spp., Phialemonium obovatum, Phialophora spp., Phoma spp., Piedraia hortai, Pneumocystis carinii, Pythium insidiosum, Rhinocladiella aquaspersa, Rhizomucor pusillus, Rhizopus spp., Saksenaea vasiformis, Sarcinomyces phaeomuriformis, Sporothrix schenckii, Syncephalastrum racemosum, Taeniolella boppii, Torulopsosis spp., Trichophyton spp., Trichosporon spp., Ulocladium chartarum, Wangiella dermatitidis, Xylohypha spp., and their synonyms. Other fungi that "obviously have pathogenic potential" (Smith, op. ct.) include, but are not limited to, Thermomucor indicae-seudaticae, Radiomyces spp., and other species of known pathogenic genera. There are also reports implicating Saccharomyces as a human pathogen (e.q., Fungemia with Saccharomycetacea, H. Nielson, J. Stenderup, & B. Bruun, Scand. J. Infect. Dis. 22:581-584, 1990). In recent years there has been a marked increase in the number of serious mycoses as a result of the growing number of immunosuppressed and immunocompromised individuals, such as transplant recipients, patients receiving chemotherapy, and HIV-infected individuals.
Fungal infection is also a significant problem in veterinary medicine including, but not limited to, candidiasis, cryptococcosis, aspergillosis, mucormycosis, pythiosis, entomophthoramycosis, oomycosis, chromomycosis, torulopsosis, infections with Penicillium spp., Trichosporon spp., Paecilomyces spp., Microsporum spp., and a variety of miscellaneous/rarer opportunistic mycoses (Opportunistic Mycoses of Man and Other Animals, J. M. B. Smith, CAB International, Wallingford, UK, 1989). Fungal infections are a common cause of nasal disease in dogs and cats (Fungal Diseases of the Nasal Cavity of the Dog and Cat, Wolf, A. M., 1992, Vet. Clin. of North Amer.:Small Anim. Prac. 22, 1119-1132). A variety of fungi, including, but not limited to, Aspergillus spp., Candida spp., Paecilomyces spp., Penicillium spp., Alternaria spp., Geotrichum spp., and Cladosporium spp., have been isolated from animal eyes and may cause fungal keratitis in several species including, but not limited to, horses, dogs, and cats (Microbiology of the Canine and Feline Eye, P.A. Gerding and I. Kakoma, 1990, Vet. Clin. of North Amer.:Small Anim. Prac. 20, 615-625). Skin infections by fungi, including, but not limited to, Microsporum canis, Trichophyton mentagrophytes, Trichophyton verucosum, Microsporum eguinum, Microsporum gallinae, and Microsporum nanum, occur in many different animals, ,both wild and domestic with some infections being specific to a given host species (Fungal Skin Infections Associated with Animal Contact, W. H. Radentz, 1991, AFP 43, 1253-1256).
Some of the fungi that infect animals can be transmitted from animals to humans. Fungal zoonotic diseases are most commonly associated with animals used as pets, with a higher frequency found among veterinary personnel owing to higher levels of contact with animals (ibid., M. R. Lappin, Vet. Clin. of North Amer. :Small Anim. Prac. 23, 57-78.). Topical and systemic antifungal agents are used to treat both humans and animals.
Fungal infections or infestations are also a very serious problem in agriculture with fungicides being employed to protect vegetable, fruit, and nut crops (F. L. McEwen and G. R. Stephenson, 1979, The Use and Significance of Pesticides in the Environment. Wiley, N.Y.). Fungicides are applied to soil, seeds, propagating material, growing plants, and produce to combat pathogens. Seed and soilborne pathogens include but are not limited to Aphanomyces spp., Armillaria spp., Cephalosporium spp., Cylindrocladium spp., Fusarium spp., Helminthosporium spp., Macrophomina spp., Magnaporthe spp., Ophiobolus spp., Phymatotrichum spp., Phytophthora spp., Pythium spp., Rhizoctonia spp., Scerotium spp., Sclerotinia spp., Thielaviopsis spp., Ustilago spp., Verticillium spp., and Whetxelinia spp., (R. Rodriguez-Kabana, P. A. Backman, and E. A. Curl, Control of Seed and Soil-Borne Plant Diseases. In Antifungal Compounds, M. Siegel and H. Sisler, eds., Marcel Dekker Inc., N.Y., 1977). Post-harvest diseases of fresh fruits and vegetable are caused by fungi including, but not limited to, Alternaria spp., Botrytis spp., Centrospora spp., Ceratocystis spp., Colletotrichum spp, Cryptoporiopsis spp., Diplodia spp., Fusarium spp., Helminthosporium spp. Monilinia spp., Nectria spp., Oospora spp., Penicillium spp., Phlyctaena spp., Phoma spp., Phomopsis spp., Rhizopus spp., Sclerotinia spp., and Verticillium spp.
It has been estimated that fungicides are employed in the growing of one-half of the world's crops (G.
Ordish and J. F. Mitchell. 1967, World Fungicide Usage. In Fungicides, an Advanced Treatise, Vol. 1, pp.39-62. D.C. Torgeson, ed. Academic Press, N.Y.) either to control disease during crop development, to improve the storage of produce, or to increase production of a particular crop. Approximately 20% of U.S. non-pasture crop land is treated with fungicides (E. W. Palm, Estimated Crop Losses Without the Use of Fungicides and Nematicides and Without Nonchemical Controls. CRC Handbook of Pest Management in Agriculture, Vol. 1, p.139f.). In economic terms, the cessation of fungicide use would result in losses to field crops, vegetable crops, and fruit and nut crops estimated to total over two billion dollars (ibid.). Some crops would be particularly hard hit, e.cr, peanut losses would be expected to be &gt;70% of the total crop, pecan losses &gt;65% of the total crop, tomato losses &gt;60% of the total crop, potato losses &gt;40% of the total crop, and fruits such as apples, cherries, peaches, and pears each &gt;50% of their total crop (ibid.).
Fungal attack of wood products is also of major economic importance with an estimated one billion dollars in damage annually (not including damage to living trees) in the U.S., even with the extensive use of existing preservatives (M. P. Levi, Fungicides in Wood Preservation, In Antifungal Compounds, M. Siegel and H. Sisler, eds., Marcel Dekker Inc., N.Y., 1977). Hundreds of fungal species have been isolated from wood products. Surface molds result from infestation by genera including, but not limited to, Trichoderma spp., Gliocladium spp., Penicillium spp., Aspergillus spp., and Alternaria spp. Sap stain fungi include, but are not limited to, Ceratocystis spp., Diplodia spp., Graphium spp., Aureobasidium spp., and Cytospora spp. Decay fungi responsible for a large proportion of the economic losses include, but are not limited to, Coniophora spp., Lentinus spp., Lenzites spp., Polyporus spp., Poria spp., and Merulius spp. Soft-rot fungi include, but are not limited to, Ascomycetes spp., Chaetomium spp., and Fungi Imperfecti.
Additional products that are susceptible to fungal infestation include textiles, plastics, paper, rubber, adhesives, emulsion polymers, leather, cosmetics, household disinfectants, deodorants, and paint. (C.C. Yeager, Fungicides in Industry, in Antifungal Compounds, M. Siegel and H. Sisler, eds., Marcel Dekker Inc., N.Y., 1977). More work has been done on paint than on any other substrate. Fungi that attack painted surfaces often disfigure the paint film to the point where replacement is required. Repainting can solve the problem only temporarily as the organism may erupt through the new coating. Paint infestations include, but are not limited to, Pullularia spp., Cladosporium spp., Aspergillus spp., and Penicillium spp. The only successful method of combating fungal growth on paint systems requires the addition of a suitable fungistat or fungicide.
The development of antifungal drug therapies has not evolved as rapidly as the development of antibacterial drug therapies in large part because the human or animal host and the fungal pathogen are both eukaryotes and have many drug targets in common. To date, most of the antifungal drugs and lead compounds have been active against components of the fungal cell surface or membrane (New Antifungal Agents, J. R. Graybill, Eur. J. Clin. Microbiol. Dis. 8:402-412, 1989; Targets for Antifungal Drug Discovery, Y. Koltin, Annual Reports in Medicinal Chemistry 25:141-148, 1989; Screening of Natural Products for Antimicrobial Agents, L. Silver & K. Bostian, Eur. J. Clin. Microbiol. Dis. 9:455-461, 1990; New Approaches for Antifunqal Drugs, P. B. Fernandes, ed, Birkhauser:Boston, 1992; Scrip's Antifungal Report, by PJB Publications Ltd, 1992). For example, polyene macrolides bind to fungal-specific ergosterol on the cell surface and azole drugs inhibit an ergosterol biosynthetic enzyme. While there has been some effort directed at intracellular targets, such as tubulin and nucleotide metabolism, the resulting compounds, such as benomyl and fluorocytosine, have problems with toxicity and resistance. Cycloheximide (Actidione) is used as a fungicide on some crops even though it is not particularly specific for fungi. Blasticidin S is also used as an antifungal agent on crops.
Not only are fungal-specific therapeutics difficult to identify, but many of the drugs currently available for treatment of mycoses have significant side effects or lack effectiveness against some important pathogens. For example, amphotericin B, an antifungal polyene macrolide antibiotic, has both short-term and long-term adverse effects, ranging from nausea and vomiting to kidney damage. Azole drugs such as clotrimazole and miconazole have such adverse side effects that their use is generally limited to the treatment of topical or superficial infections. The more recently developed triazole drugs, such as fluconazole, have fewer side effects but are not completely effective against all pathogens. Also, some evidence exists for the development of resistance to these drugs. There is therefore an ongoing need for novel antifungal drugs with few side effects and with effectiveness against pathogens for which current drugs are inadequate.
Furthermore, fungal and mycotic pathogens often are either naturally resistant, or develop resistance, to many therapeutics by virtue of cellular permeability barriers to drug entry. Development of fungicide resistance occurs when a fungal cell or a fungal population that originally was sensitive to a fungicide becomes less sensitive by heritable changes after a period of exposure to the fungicide. Most instances of resistance are related to a change at the site of action or a change in the uptake of the fungicide, with detoxification being a rare event (J. Dekker, Preventing and Managing Fungicide Resistance, Pesticide Resistance: Strategies and Tactics in Man). In certain applications (e.g., agriculture) it is possible to combat resistance through alternation of fungicides or the use of fungicide mixtures. To prevent or delay the buildup of a resistant pathogen population, different chemicals that are effective against a particular disease must be available. One way of increasing the number of available chemicals is to search for new sitespecific inhibitors (ibid.). Thus, the challenge is to develop methods for identifying compounds which can penetrate the pathogen and specifically kill it or arrest its growth without also adversely affecting the human, animal, or plant host.
Classical approaches for identifying antifungal compounds have relied almost exclusively on inhibition of fungal growth as an endpoint. Libraries of natural products, semisynthetic, or synthetic chemicals are screened for their ability to kill or arrest growth of the target pathogen or a related nonpathogenic model organism. These tests are cumbersome and provide no information about a compound's mechanism of action. The promising lead compounds that emerge from such screens must then be tested for possible toxicity to the human, animal, or plant host, and detailed mechanism-of-action studies must subsequently be conducted to identify the affected molecular target and precisely how the drug interacts with this target.
Because mycoses are assuming even greater clinical importance, especially with the growing number of immunocompromised or immunosuppressed individuals, pressure has mounted to develop more effective methods for antifungal and antimycotic drug discovery. One approach uses different in vitro assays to target specific pathways that are deemed either to be unique to fungi, or sufficiently different from their human, animal, or plant counterparts that one might reasonably expect the fungal pathway to be differentially sensitive to the desired drug. Examples of pathways that are unique to fungi include chitin synthesis and degradation. Individual enzymes responsible for key steps in these pathways are being purified and used for in vitro studies to identify potential inhibitors. Examples of fungal targets that might be differentially sensitive to a drug compared to their human, animal, or plant counterparts include components required for mRNA splicing and topoisomerases. The specific molecular targets can be purified and used for in vitro studies to identify potential inhibitors. The in vitro studies in use are of two broad types: 1) purified target macromolecules are used in in vitro assays to screen large compound libraries for inhibitory drugs, or 2) the purified target molecule is used for a rational drug design program which requires first determining the structure of the macromolecular target or, preferably, the structure of the macromolecular target in association with its customary substrate or ligand. This information is then used to design inhibitory compounds which must be synthesized and tested further. Test results are used to refine the molecular models and drug design process in an iterative fashion until a lead compound emerges.
While these current methods offer certain improvements over the traditional screens that simply evaluate fungal growth in the presence and absence of a test compound, they still have limitations. On the positive side, these methods represent a relatively efficient, focused approach to drug discovery and the lead compounds they identify, by definition, will have known targets and mechanisms of action. However, because these methods are performed in vitro using a purified macromolecular target, the lead compounds that emerge may fail to kill or arrest the growth of fungal pathogens for a variety of reasons. The potential lead may not get into the fungal cell because of transport or permeability barriers. If it does get into the cell it may be inactivated by sequestration, modification or degradation. Conceivably, the cell may have a redundant biochemical pathway or a target that is not sensitive to the drug. Also, the theoretical basis for selecting a single macromolecule as the target for an in vitro drug development program may rest on assumptions that later prove unwarranted.
It has been recognized by several authors that the fungal translational elongation factor EF-3 would be a good target for antifungal compounds. However, as is clear from the following citations, none of these authors have suggested specific m ds for exploiting EF-3 to identify new anti-fungal or anti-mycotic agents. M. F. Tuite, Trends in Biotechnol. 10:235-239, 1992, describes the identification and exploitation of new antifungal targets. He states that:
"EF-3 is an absolute requirement for protein synthesis on S. cerevisiae ribosomes but not on the mammalian ribosome. PA1 "EF-3 therefore represents an almost unique example of an essential polypeptide apparently unique to fungal species yet which has no apparent mammalian counterpart (although the essential activity EF-3 supplies to fungal ribosomes actually may be an intrinsic property of a mammalian ribosomal protein). The demonstration of its essential nature in S. cerevisiae highlights the potential of EF-3 as a target for rationally designed antifungal drugs. While inhibition of the ribosome-dependent nucleotidase activity associated with EF-3 may not represent an effective target, an ability to block its association with the ribosome may be a more realistic goal. The demonstration that EF-3 from an important human pathogenic yeast, namely C. albicans, can be functionally expressed in a genetically manipulable host such as S. cerevisiae will greatly assist a molecular genetic dissection of the functional role of this translation factor in protein synthesis and thereby facilitate attempts to rationally design antifungal agents targeted at EF-3." citations omitted.! Colthurst et al., 80 FEMS Microbiology Letters 45, 1991 states PA1 "EF-3 may also represent an important potential target for anti-fungal agents particularly given the increasing prevalence of Candida infections amongst individuals with suppressed immune systems."
Subsequent studies have confirmed that soluble EF-3 is found only in fungi. . . . While its precise role in translation remains to be defined, biochemical studies have suggested that EF-3 provides an essential nucleotidase activity. . . . Preventing EF-3 binding to the fungal ribosome may therefore represent a new antifungal strategy and, while studies to date have focused on EF-3 from S. cerevisiae, the recent isolation and demonstration that C. albicans EF-3-encoding gene can substitute functionally for its S. cerevisiae counterpart, will provide a means of bypassing the difficulties of undertaking molecular-genetic studies in C. albicans. . . Identification of a potential antifungal target, however, is only the first step in ultimately producing an effective antifungal compound to combat the increasing occurrence of life-threatening fungal diseases. Either (1) a high through-put screen must then be developed to identify potential inhibitors that act specifically on the target, or (2) detailed structural information must be obtained for the target molecule to facilitate the rationale design of effect antifungal drugs. These are not trivial tasks, and they both rely on the identification of new antifungal targets." citations omitted.! Colthurst et al., Mol. Microbiol. 6:1025, 1992 state: